During the past year, we have used a positive immunoselection method to isolate a Thy-1 positive revertant of the Thy-1 negative Rlo1-4 line. The technique makes use of the ability of surface-bound antigen-antibody complexes containing the enzyme catalase to protect cells from the toxic effects of hydrogen peroxide. The revertant cells, which apparently exist at a frequency of approximately 10-7, produce a Thy-1 molecule indistinguishable from the original parent line andresemble that line in being far less tumorigenic than the Thy-1 negative variant. We also have succeeded in growing several lines of alloreactive normal T cells. BALB/c spleen cells obtained from mice immunized against C57Bl/6 cells have been grown in IL-1-supplemented media for 3 months and four "clones" have been isolated by limit dilution cloning. We are now typing the clones for a variety of surface markers. Preliminary results indicate them to be Thy positive, H-2KD posi-tive, TL negative and Ly-1.1 positive. Finally, the murine T-cell alloantigen, Thy-1.2, has been transferred into a Thy-1.1-bearing mouse T-cell lymphoma, BW5147, using DNA extracted from BALB/c thymocytes (Thy-1.2-expressing cells) and the calcium phosphate precipitation method fo DNA-mediated gene transfer. Two weeks later, they were reanalyzed for the number of "stable" transformants expressing Thy-1.2. Eight percent (0.1% of the original population) of the cells of the planned population and 5% of the sorted population were stained, as compared to 2% of the control. In one experiment, the 8% positive population was resorted and was subsequently found to be 28% positive one month later. Now, 4 months have passed and it is still 28% positive. During the next year, we shall (1) attempt to prepare monoclonal antibodies which mimic the effects of anti-RLo1 antibodies; (2) continue to examine the efficacy of DNA-mediated gene transfer a means of introducing novel cell surface markers into differentiated T cell clones; and (3)\continue our studies on antigen-dependent IL-2 secretion by alloreactive T cell clones. When these cells are sufficiently well-characterized, we shall prepare variants of them and study the effect of alterations in surface phenotype on antigen-dependent reactivity.